The DNA Sensor IFIX Drives Proteome Alterations To Mobilize Nuclear and Cytoplasmic Antiviral Responses, with Its Acetylation Acting as a Localization Toggle.
The DNA Sensor IFIX Drives Proteome Alterations To Mobilize Nuclear and Cytoplasmic Antiviral Responses, with Its Acetylation Acting as a Localization Toggle.
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DOI：
10.1128/msystems.00397-21
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发表时间：
2021-06-29
期刊：
影响因子：
6.4
通讯作者：
Cristea IM
中科院分区：
文献类型：
--
作者：
Howard TR;Crow MS;Greco TM;Lum KK;Li T;Cristea IM
DNA传感器是先天免疫的关键组成部分，使细胞能够识别病原体的DNA基因组感染。干扰素诱导蛋白X （IFIX）是PYHIN蛋白家族的一员，是一种DNA传感器，能够在细胞核或细胞质内与双链DNA （dsDNA）结合后促进免疫信号传导。在这里，我们研究了外源DNA引入细胞核或细胞质时IFIX对细胞蛋白质组的影响，以及控制IFIX亚细胞定位的调控中心。使用定量质谱法，我们确定了crispr介导的IFIX敲除对成纤维细胞核和细胞质蛋白质组的影响。在单纯疱疹病毒1型（HSV-1）感染期间，检测蛋白质组对核病毒DNA的响应，或者在转染来自牛痘病毒（VACV 70-mer）的dsDNA后，检测细胞质病毒DNA的响应。我们发现IFIX广泛影响核和细胞质蛋白质组，诱导免疫信号、DNA损伤反应和囊泡介导转运蛋白丰度的改变。为了表征IFIX在DNA传感过程中调控其定位的特性，我们进行了删除和诱变实验。我们发现IFIX包含一个多部核定位信号（NLS），并突出了其核定位的主要贡献基序。通过免疫亲和纯化，我们确定了IFIX的乙酰化和磷酸化位点。乙酰化或电荷模拟物的突变表明，位于NLS内的K138乙酰化影响核定位。总之，我们的研究建立了一种调节IFIX亚细胞定位的机制，并将这种定位与IFIX参与宿主细胞对致病性DNA的反应联系起来。哺乳动物细胞必须能够检测并对入侵的病原体作出反应，以防止感染的传播。DNA传感器，如IFIX，是结合病原体来源的双链DNA并诱导抗病毒细胞因子表达的蛋白质。在这里，我们描述了在病毒感染和DNA转染期间需要IFIX的宿主蛋白质组变化。我们发现IFIX在细胞核和细胞质内调动了许多途径和蛋白质组改变，指出了一种在免疫信号、DNA损伤反应和转录调节中发挥作用的多功能蛋白。接下来，我们研究了核定位所需的IFIX结构域，发现其通过多部分核定位基序进行调控。该基序的乙酰化促进了IFIX的细胞质定位，这与它在细胞核和细胞质中检测致病DNA一致。本研究确定了NLS乙酰化是调节PYHIN蛋白家族核DNA传感器定位的保守机制。
DNA sensors are critical components of innate immunity that enable cells to recognize infection by pathogens with DNA genomes. The interferon-inducible protein X (IFIX), a member of the PYHIN protein family, is a DNA sensor capable of promoting immune signaling after binding to double-stranded DNA (dsDNA) within either the nucleus or cytoplasm. Here, we investigate the impact of IFIX on the cellular proteome upon introduction of foreign DNA to the nucleus or the cytoplasm as well as regulatory hubs that control IFIX subcellular localization. Using quantitative mass spectrometry, we define the effect of CRISPR-mediated IFIX knockout on nuclear and cytoplasmic proteomes in fibroblasts. Proteomes are probed in response to either nuclear viral DNA, during herpes simplex virus 1 (HSV-1) infection, or cytoplasmic viral DNA, following transfection with dsDNA derived from vaccinia virus (VACV 70-mer). We show that IFIX broadly impacts nuclear and cytoplasmic proteomes, inducing alterations in the abundances of immune signaling, DNA damage response, and vesicle-mediated transport proteins. To characterize IFIX properties that regulate its localization during DNA sensing, we perform deletion and mutagenesis assays. We find that IFIX contains a multipartite nuclear localization signal (NLS) and highlight the main contributing motif for its nuclear localization. Using immunoaffinity purification, we identify IFIX acetylation and phosphorylation sites. Mutations to acetyl or charge mimics demonstrate that K138 acetylation, positioned within the NLS, affects nuclear localization. Altogether, our study establishes a mechanism regulating IFIX subcellular localization and contextualizes this localization with the involvement of IFIX in host cell responses to pathogenic DNA. IMPORTANCE Mammalian cells must be able to detect and respond to invading pathogens to prevent the spread of infection. DNA sensors, such as IFIX, are proteins that bind to pathogen-derived double-stranded DNA and induce antiviral cytokine expression. Here, we characterize the host proteome changes that require IFIX during both viral infection and DNA transfection. We show IFIX mobilizes numerous pathways and proteome alterations within the nucleus and the cytoplasm, pointing to a multifunctional protein with roles in immune signaling, DNA damage response, and transcriptional regulation. We next interrogate the IFIX domains required for nuclear localization, discovering its regulation via a multipartite nuclear localization motif. The acetylation of this motif promotes IFIX cytoplasmic localization, in agreement with its detection of pathogenic DNA in both the nucleus and the cytoplasm. This study established NLS acetylation as a conserved mechanism for regulating the localization of nuclear DNA sensors from the PYHIN family of proteins.